JPH1077236A - Stabilizing additive in water-soluble medicine preparation of digestive enzyme mixture containing lipase and protease, preparation containing the same additive and kit for producing the same digestive enzyme mixture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound soluble in an aqueous medium, stable for a long period of time, stable to loss of fat decomposing activity under action of water, suitable as an aqueous solution to continuously flow into a mammalian gastrointestinal region. SOLUTION: A complex lipid uses an anion salt of the formula (R<1> and R<2> are each 10-25C alkanoyol, etc.; R<3> is H, carboxyl, etc.; R<4> is H or 10-25C hydrocarbon chain; A is O or NH) as a stabilizing additive for a digestive enzyme containing a lipase, etc. For example, a vegetable lecithin of a natural plant such as soybean is preferably and a complex lipid as a mixture of various lipids is suitably. In producing and processing a medicine preparation, the complex lipid is added, deactivation of lipase is prevented, lipid decomposition activity during a process is advantageously stabilized, a complex fat depends on a pH value in the production and the pH value is usually 3.5-9.0, preferably 4.0-7.0, especially preferably 5.0-6.5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the action of water, which contains protease / lipase mixtures, in particular pancreatin, and which is suitable for the production of aqueous solutions for continuous introduction into the gastrointestinal tract by means of a probe. The invention relates to the use of complex lipids as additives to water-soluble pharmaceutical preparations of digestive enzyme mixtures, which are stabilized against a reduction of the lipolytic activity below. Furthermore, the present invention is stabilized by a mixture of lipase and protease-containing digestive enzymes, in particular complex lipids, against the reduction of lipolytic activity under the action of water and can be introduced into the gastrointestinal tract of mammals or humans using probes. It relates to a water-soluble pharmaceutical preparation of a pancreatin-containing digestive enzyme mixture which is suitable for the production of an aqueous solution.

[0002] In mammals, especially humans, there may be manifestations of digestive enzymes caused by pathological changes in the pancreas, for example due to chronic pancreatitis, indigestion after gastric surgery, liver or gallbladder disease. Such deficiency symptoms may optionally still contain lipase additives, such as pancreatic juice enzymes,
It is already known that treatment can be effected, in particular, by administration of a non-living pancreatin-containing digestive enzyme mixture such as pancreatin. Pancreatic juice enzymes are usually administered orally in the form of a solid preparation. In the case of oral administration, the enzyme mixture is coated with a gastric juice in order to prevent the administered enzyme mixture from being irreversibly altered by undesired gastric acids and proteolytic enzymes such as pepsin already present in the stomach. It is necessary to provide. Such a coating allows the enzyme mixture to pass intact through the stomach to its site of action, the duodenum, where the neutral or slightly alkaline conditions that predominate there cause the protective layer to collapse and the enzyme Is released. Like the pancreatic enzymes specific to the body of healthy humans, orally supplied enzymes then exert their enzymatic action, in particular starch hydrolyzing, lipolytic and proteolytic activities.

[0003] Such solid pancreatin preparations which can be coated with a gastric juice-resistant film in the form of micropellets are described, for example, in German Patent (DOS) 4227385.4.

For patients with dyspepsia, for example bedridden patients with long-lasting dyspepsia such as chronic pancreatic insufficiency, liquids may be used instead of solid dosage forms, for example by continuous application using a sonde. It is desirable to administer the non-native endogenous digestive enzymes over a long period of time.

[0005] Pancreatin-containing digestive enzyme mixtures, in particular liquid dosage forms of pancreatin, have hitherto not been available because liquid aqueous formulations of such enzyme mixtures are not stable over time. . In particular, it is evident that the proteolytic action of proteases likewise contained in the mixture, such as trypsin or chymotrypsin in the presence of water, rapidly reduces the activity of the lipase contained in the mixture. Thus, external conditions (temperature, pH value) can cause a significant loss of lipase activity within a short time in aqueous pancreatin preparations.

In order to be suitable for continuous introduction into the gastrointestinal tract by application with a probe, an aqueous solution of a digestive enzyme mixture containing lipase and protease, in particular a mixture of digestive enzyme containing pancreatin, can be used for many hours. For example, it must be stable over a period of 8 hours. In particular,
The solution must not contain or contain particles that occlude the sound. The main requirement for such a solution is that the highest and uniform activity of all digestive enzymes contained therein is maintained over the entire administration period. Furthermore, a solution suitable for continuous gastrointestinal application requires that this solution has no bacterial growth and thus can be protected, for example, against bacterial growth, and in particular be able to be sterilized.

[0007]

Accordingly, an object of the present invention is to provide a lipase and a lipase which are stable in aqueous media for a long time and which are stabilized against the loss of lipolytic activity under the action of moisture. It was to provide a pharmaceutical formulation of a protease-containing water-soluble digestive enzyme mixture.

[0008]

The object of the present invention is to provide a mixture of lipase and protease-containing digestive enzymes, in particular pancreatin-containing, which is suitable for the production of an aqueous solution for continuous introduction into the gastrointestinal tract using a probe. The use of a complex lipid as a stabilizing additive against the reduction of lipolytic activity under the action of water in a water-soluble pharmaceutical formulation of a digestive enzyme mixture. Furthermore, the subject of the present invention is a water-soluble pharmaceutical preparation thus stabilized of a digestive enzyme mixture. Similarly, the subject of the present invention is a kit for producing an aqueous solution of a digestive enzyme suitable for a continuous sonde application.

[0009] Complex lipids suitable as stabilizing additives according to the invention are generally insoluble in acetone. In particular,
Phospholipids with and without carbohydrates and glycolipids with and without carbohydrates and mixtures thereof are counted. Advantageously, only phospholipids or mixtures containing phospholipids and glycolipids are used.

The phospholipids which can be used according to the invention as stabilizing additives for lipase and protease-containing digestive enzyme mixtures, in particular pancreatin-containing digestive enzyme mixtures, have the general formula I

[0011]

Embedded image

Wherein R ¹ is hydrogen or its hydrocarbon radical may optionally contain from 1 to 4 double bonds,
Represents an alkanoyl group having 5 carbon atoms, R ²
Represents an alkanoyl group having 10 to 25 carbon atoms, wherein hydrogen or a hydrocarbon group thereof may optionally contain 1 to 4 double bonds, or when R ¹ does not represent hydrogen R ³ may be hydrogen, amino, lower trialkylammonium, a lower alkyl group optionally substituted by a carboxyl group bonded to a carbon atom having an amino function or a cycloalkyl group substituted by hydroxy. Wherein R ⁴ may contain hydrogen or optionally 1 to 4 double bonds, 10 to 10
Represents a hydrocarbon chain having 25 carbon atoms and A represents oxygen or NH]. Salts of anions with physiologically acceptable cations are suitable.

The physiologically acceptable cations include ammonium ions, alkali or alkaline earth metal cations, especially sodium, potassium or calcium, and other physiologically acceptable monovalent or polyvalent cations. Is appropriate. As long as R ³ contains a nitrogen atom, this nitrogen atom can form a quaternary ammonium ion, which can also serve as a cation, thus forming an outwardly uncharged inner salt. Is done.

The radicals R ¹ and / or R ^{2 in} the compounds of the formula I
Represents an alkanoyl group, which may be straight-chain or branched, usually unbranched and have 10 to 25, in particular 16 to 20, carbon atoms. Optionally, the alkanoyl group has up to four double bonds. Alkanoyl groups can be, in particular, groups of long-chain fatty acids such as nervonic acid, lignoceric acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid or arachidonic acid.

As long as the substituent R ³ represents or contains a lower alkyl group, said alkyl group may be straight-chain or branched and in particular has 1 to 4, preferably 1 to 2 carbon atoms. Can be contained. As long as a cycloalkyl group R ³ is substituted by hydroxy, the cycloalkyl group may be substituted once or several times by inclusion to and hydroxy 3 to 6 carbon atoms. Preferably, a cycloalkyl group contains 5 to 6 carbon atoms, each of which may be substituted by hydroxy.

The radical R ³ O preferably represents hydroxy or an alkoxy radical resulting from the esterification of a monohydric or polyhydric alcohol with a phosphate group,
In this case, the monohydric or polyhydric alcohol is aminoethanol,
It is selected from the group consisting of choline, serine, glycerin and myo-inositol.

As long as the radical R ⁴ represents a hydrocarbon chain, this hydrocarbon chain may be straight-chain or branched, is usually unbranched and has 10 to 25, preferably 12
It contains from 20 to 20, particularly preferably 15 carbon atoms. Optionally, there are up to 4 hydrocarbon chains, preferably 2
And particularly preferably one double bond.

The group A can represent an oxygen or NH group.

Preferably, phosphatidic acid (1,2-diacyl- sn -glycerol-3-phosphate) and phosphatidylcholine (1,2-diacyl- s ) are used as phospholipids.
n -glycerol-3-phosphorylcholine), phosphatidylethanolamine (1,2-diacyl- sn -glycerol-3-phosphorylethanolamine), phosphatidylserine (1,2-diacyl- sn -glycerol-3-phosphorylserine) and phosphatidyl Inosit (1,2-diacyl-Sn-glycerol-phosphoryl inosit); when the phospholipid is of animal origin, such as chicken eggs, sphingomyelin, as well as mixtures of these compounds. Can be The aforementioned 1,2-diacyl phospholipids can be partially hydrolyzed under certain conditions, for example under the enzymatic action of phospholipase. At that time, the nature of the phospholipase, group R ^1, R ^2, R ³ or 1,2-diacyl lipid [R ³ OPO _2] ^- can also be replaced by hydrogen. Hydrolysis of at least one of the above molecular residues per phospholipid molecule results in so-called Lyso phospholipids, in particular lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinosit, lysophosphatidylserine and lysophosphatidic acid. . These phospholipids are also suitable as stabilizing additives for lipase and protease-containing digestive enzyme mixtures in the sense of the present invention, in particular pancreatin-containing digestive enzyme mixtures.

Glycolipids, which appear especially in plants,
General formula II

[0021]

Embedded image

In which R ⁵ and R ⁶ independently of one another are each an alkanoyl radical having 10 to 25 carbon atoms or a hydrogen radical in which the hydrocarbon radical can optionally contain 1 to 4 double bonds. Wherein R ⁵ and R ⁶ cannot simultaneously represent hydrogen, and R ⁷ is selected from the group whose sugar component is D-fructosyl, D-galactosyl, D-glucosyl and D-mannosyl. Represents a mono- or disaccharide residue], a so-called plant glycolipid (P
hyglycolipids) as well as mixtures thereof can be used.

As long as R ⁵ and R ⁶ in the compound of the formula II represent an alkanoyl group, this alkanoyl group is branched or unbranched, usually unbranched and 10 to 25, in particular 16 to Contains 20 carbon atoms. Optionally, the alkanoyl group can contain up to four double bonds. As alkanoyl groups, particularly, palmitic acid, palmitoleic acid, stearic acid,
Examples include groups of long chain fatty acids such as oleic, linoleic, linolenic or arachinic acids.

The group R ⁷ is a sugar molecule D-galactose, D-galactose.
It represents a monosaccharide or disaccharide residue which may be composed of glucose, D-mannose or D-fructose.
Particularly preferably, R ⁷ is D-galactose (which is monogalactosyl-diglyceride, MGDG) or digalactose (which is digalactosyl-diglyceride, DGDG; 1,2-diacyl- [α-D-galactosyl- (1 → 6) -β-D-galactosyl- (1 →
3)]- sn -glycerin).

The phospholipids of the general formula I and the glycolipids of the general formula II are in each case added to the central carbon atom of the glycerin matrix by 1
It may have a plurality of chirality centers, and may have an R configuration or an S configuration. For the purposes of the present invention, formula I and / or formula I
The individual stereoisomeric forms of the compounds of I as well as the corresponding mixtures can be used.

The stabilization according to the invention of a pharmaceutical preparation of a lipase and protease-containing digestive enzyme mixture, in particular a pancreatin-containing digestive enzyme mixture, depends on various phospholipids and, if appropriate, those which can be produced from natural products. Lipid mixtures, which are mixtures of various glycolipids, have proven to be advantageous. A preferred example of such a natural lipid mixture is lecithin. The source of such natural lecithins may in particular be plants such as soybeans, sunflowers, rapeseed, corn or peanuts, as well as animals or animal products such as yolk or brain material, and also microorganisms.
Naturally occurring lecithin may be purchased commercially from a variety of sources.

Among the vegetable lecithins, in particular soy lecithin, in particular phospholipid-enriched soy lecithin such as, for example, soy lecithin having a phospholipid content of about 98%, are suitable for the purposes of the present invention. Untreated, non-enriched plant lecithin usually contains a certain amount of plant glycolipids. Naturally occurring lecithin is a mixture of various phospholipids, and if native to plants, glycolipids are also present and their composition is not unitary and can vary depending on their source. Thus, in addition to the components described above, still other phospholipids may be included in secondary amounts. Table 1 serves to illustrate the average composition of some untreated, non-concentrated commercial natural lecithin.

[0028]

[Table 1]

The pharmaceutical preparations stabilized according to the invention contain in particular a pancreatin-containing digestive enzyme mixture.

In the context of the present invention, pancreatin refers to pancreatin isolated from the mammalian pancreas, the active protease content of which is optionally determined by the autolytic degradation of the protease zymogen originally contained therein. Increased.

Preferably, the pancreatin-containing digestive enzyme mixture in the pharmaceutical preparation stabilized according to the present invention is a mixture of various digestive enzymes produced from the pancreas of a mammal, pancreatin, in particular, pig pancreatin. It may be creatine. Mammalian pancreatin suitable as a digestive aid in human nutrition, especially pancreatin from porcine pancreas, does not always contain lipase in sufficient amounts for human needs. Thus, for example, a lipase produced from a microorganism can be added to such a pancreatin preparation. Pancreatin obtained in this way /
Mixtures of lipases are also suitable enzyme mixtures.

In pancreatin isolated from mammals, if pancreatin has not been subjected to other pretreatments, the protease is usually present in the form of a proteolytically inactive precursor zymogen, mostly. . Thus, for pharmaceutical purposes, it may be advantageous to still carry out a hydrolysis treatment (autolysis) on the crude pancreatin obtained from the pancreas by a suitable precipitation method in a manner known per se. In this process, the zymogen is turned into an active protease. In this autolytically pretreated pancreatin (abbreviated below as F-pancreatin), these F-pancreatins are dangerous with respect to lipolytic activity, especially because of the high content of active proteases. Has been exposed. The use of the complex lipids according to the invention is particularly suitable for stabilizing lipolytic activity in pharmaceutical preparations containing F-pancreatine. Here, the stabilization of the lipase activity is effected by the inactivation of the active proteases contained in the mixture, so that the enzyme mixture thus stabilized has amylolytic and lipolytic activities, as well as proteolytic activities. That is phenomenal.

The pancreatin-containing digestive enzyme mixture in the pharmaceutical preparation to be protected according to the invention may contain, in addition to pancreatin, a lipase such as that contained in plants or microorganisms. Lipases from microorganisms can be bacterium or, for example, strain Rhizopus.
And those produced from filamentous fungal cultures such as mold fungi.

As an additional protease component, the pancreatin-containing digestive enzyme mixture in the pharmaceutical preparations to be protected according to the invention can be used in addition to other animal and plant proteases and, in particular, molds of microorganisms such as bacteria or, for example, the strain Aspergillus. A protease that can be produced from a culture of a filamentous fungus such as a fungus can be added.

The pancreatin-free digestive enzyme mixture may contain lipase from plant or microbial tissue as a lipase. The lipase from the microbial tissue may be as produced from a bacterium or a fungal culture such as, for example, a fungus of the genus Rhizopus. The proteases contained in the pancreatin-free digestive enzyme mixture can be produced from animal or plant proteases, and in particular from microorganisms, such as bacteria or fungal cultures such as molds of the strain Aspergillus, for example. Proteases.

The pharmaceutical composition according to the invention may additionally contain, in addition to the digestive enzyme mixture and the complex lipid, water-soluble pharmaceutical auxiliaries and / or additives. For example, a carrier substance such as carbohydrates, for example mannitol, or soluble proteins and preservatives may be included.

Pharmaceutical preparations of the pancreatin-containing digestive enzyme mixture in the sense of the present invention can be used to stabilize lipolytic activity under the action of water in addition to pancreatin, especially F-pancreatin. It may be a water-soluble powder which contains a sufficient amount of water-soluble auxiliaries and / or additives which are known per se.

For the production of the water-soluble powder, the water-insoluble components can be sufficiently removed from the enzyme mixture. For this purpose, the aqueous preparations of F-pancreatin are separated from the insoluble solids by suitable methods known per se for the separation of solids, for example by centrifugation or filtration, and the resulting solution is optionally treated with other aids. After the addition of the agents and / or additives, they can be sterilized by methods known per se, for example by means of sterile filtration. The resulting clear, optionally sterile solution content can then be obtained again as a solid by known drying methods, for example by freeze-drying. The formulation thus obtained is suitable for continuous and uniform application to the gastrointestinal tract of a patient, for example by means of a probe, and is stable for many hours, for example up to 8 hours.
It is optionally suitable for the production of a solution without bacterial growth.

Continuous application refers to the administration of an optionally sterile aqueous solution containing a pharmaceutical preparation according to the invention, for about 1 hour up to many hours, for example up to 8 hours, almost continuously, almost overnight. . The continuous application of the solution can advantageously be effected by a probe inserted in the gastrointestinal tract, for example in the stomach or small intestine.

The effectiveness of lecithin addition is sufficiently independent of the relative lipase / protease ratio in the enzyme mixture. For example, the lipase / total protease ratio— “F
edition International
Pharmaceutical "(hereinafter abbreviated as FIP), the ratio of the respective activities (R. Ruyssen and A. Lauwers, Ph.
armaceuticalEnzymes, Scien
Tifty Publishing Company,
Gent 1978, pp. 74-82, below, quoted as "Lauwers") and described below in relative activity units (abbreviated FIP-E / g)-from about 5: 1 to about 30. A mixture of enzymes which can be 1: 1 is suitable.

In principle, the addition according to the invention of the complex lipids to the pharmaceutical preparation of a digestive enzyme mixture containing lipase and protease, in particular a digestive enzyme mixture containing pancreatin, stabilizes against a reduction in lipolytic activity under the action of water. Cause. Moisture generally refers to an aqueous liquid that can reach from the very low water content of the digestive enzyme mixture powder to an aqueous formulation of this powder.

The addition of the complex lipids according to the invention can result in the inactivation of lipases already during the production and processing of the pancreatin-containing digestive enzyme mixture used in the pharmaceutical preparation, for example by wet treatment of the enzyme mixture. It has been found to be advantageous for stabilizing the lipolytic activity during such steps of the production or production process.

The stabilizing effect of added complex lipids, in particular lecithin, in aqueous solutions of pharmaceutical preparations containing digestive enzyme mixtures depends, inter alia, on the predominant pH value in the preparation. According to the invention, a pH in the range of 3.5 to 9.0, preferably pH
Within the range of 4.0 to 7.0, particularly preferably pH 5.0.
A pH value in the range of ６6.5 has been found to be advantageous.

In order to obtain a significant stabilization of the lipolytic activity in the water-soluble pharmaceutical preparation of the digestive enzyme mixture and in the aqueous solution for the sonde application which can be produced from this preparation, a certain minimum amount of complex lipid is added. It is necessary. Usually, the digestive enzyme mixture used in the formulations according to the invention contains only lipase from mammalian pancreatic secretions, for example 2000 to 20000 FIP-E / g and lipase from microorganisms alone or from the pancreas. 2000 to 50000 when contained in combination with lipase
It may have a lipase content expressed in activity units of 0FIP-E / g. At least 1% by weight of complex lipid based on the amount of digestive enzyme mixture containing solid pancreatin
Amounts, for example from 1 to 10% by weight, are suitable for obtaining significant stabilization. The addition of larger amounts of complex lipids is likewise possible, but no longer causes a significant improvement in the stabilization of the lipolytic activity. Thus, for example, in order to stabilize pancreatin or a mixture containing pancreatin and additional lipase with complex lipids, in particular lecithin, at least 1% by weight of lecithin, preferably 2 to 5% by weight, particularly preferably about An addition of 3% by weight is appropriate.

With the use according to the invention of the complex lipids, an aqueous solution, which can be produced from a pharmaceutical preparation of a mixture of digestive enzymes containing pancreatin, which has a tendency to have a rapid decrease in lipolytic activity, The lipolytic activity can be stabilized so that it hardly decreases over a long period of time. Therefore, F stabilized by the addition of lecithin
-An aqueous solution of pancreatin has a lipolytic residual activity of 85% of its original activity after an incubation time of 8 hours at room temperature. In aqueous F-pancreatin stabilized by complex lipids, 50% of the lipolytic activity can still be demonstrated even after a 24-hour incubation period. In contrast, in the unstabilized comparative solution, under the same conditions, the lipolytic activity dropped to less than 20% of the original activity after 8 hours.

The stabilization of the water-soluble pharmaceutical preparation of the lipase and protease-containing digestive enzyme mixture against the reduction of the lipolytic activity in an aqueous medium according to the invention allows the patient to continuously feed such a digestive enzyme mixture in the form of an aqueous solution. Discloses the possibility of feeding into the gastrointestinal tract. Of course, the aqueous solution used for this should have no bacterial growth and, in particular, should be rather sterile. Solutions without bacterial growth are, for example,
It may be a solution in which the growth of bacteria capable of self-propagation has been inhibited by the addition of a preservative.

In accordance with the present invention, there is provided a method for preparing an aqueous solution without bacterial growth of a digestive enzyme mixture stabilized against reduced lipolytic activity, suitable for continuous introduction into the gastrointestinal tract by a sonde. A kit is provided which comprises as components: a) a lipase and protease containing digestive enzyme mixture, which may optionally contain a sufficient amount of complex lipids to stabilize the aqueous solution to be produced against a reduction in lipolytic activity. Pharmaceutical preparations without bacterial growth of water-soluble solids, and b) to be produced in solid pharmaceutical preparations which can contain physiologically acceptable salts and auxiliaries in addition to water and which are mentioned under a) If the complex lipid is not contained in an amount sufficient to stabilize the lipolytic activity of the aqueous solution, it is necessary to stabilize the aqueous solution to be additionally produced against the lipolytic activity. Containing a sufficient amount of complex lipids, characterized in that it contains an aqueous solvent having no bacterial growth in an amount sufficient for the preparation of an aqueous solution.

In particular, the solid preparations used in the kit a)
May be a lyophilized lipase and protease containing digestive enzyme mixture, optionally containing complex lipids. Preferably, the digestive enzyme mixture is a pancreatin-containing digestive enzyme mixture.

An aqueous solution having no bacterial growth can be obtained by addition of a preservative known per se, for example, paraben. Similarly, a sterile solution which is a solution having no bacterial growth can be obtained by a sterilization method known per se, for example, sterile filtration.

The lipids contained in the kit can be present, especially as additives already contained in the digestive enzyme mixture (component a)). In order to produce a powder of the digestive enzyme mixture, which already contains the complex lipid, for example, a solution of the complex lipid optionally without bacterial growth is mixed with a solution of the digestive enzyme mixture optionally without bacterial growth, and subsequently Drying can be performed by a method known per se, for example, freeze-drying. In order to obtain a solution that can be applied by the probe, the powder containing the complex lipid and the digestive enzyme mixture must be mixed with the solvent also contained in the kit under conditions that are low in bacteria or sterile. No. However, the complex lipid may already be present in the solvent (component b)), for example, dissolved as a colloid. In order to obtain a solution which can be applied by the probe, the aqueous solution or colloid containing the complex lipids in this case must be mixed, optionally under sterile conditions, with the digestive enzyme mixture (component a)).

[0051]

【Example】

1. Fat content of pancreatin aqueous solution with soy lecithin
Corresponding samples were prepared and incubated at 30 ° C. to determine the different changes in lipolytic activity with and without the addition of complex lipids in the stabilized aqueous pancreatin preparation. From the incubated sample,
The change in lipase activity with time is referred to as “Federati
on Internationalale Pharmac
eutique / European Pharmaco
peia "(abbreviated below as FIP / Ph. Eur., L
auers, pp. 74-82). In this standard assay, a sample whose lipase activity is to be determined was allowed to act on olive oil triglycerides under hydrolysis conditions, and the carboxylic acid liberated was titrated with sodium hydroxide to a pH value of 9. The lipase activity of the sample is determined by comparing the rate at which the sample hydrolyzes the olive oil emulsion with the rate at which the suspension of the pancreatic reference powder hydrolyzes the same substrate under the same conditions.

1.1 Test of lipase stability without lecithin addition 79.35 mg of water-soluble, lyophilized F-pancreatin with a lipolytic activity of 50473 FIP-E / g
With ice-cooled pure water (Nano from Barnstead)
pur ^(R) ) was dissolved in 4.0 ml, and the pH value was adjusted to 1NH.
The mixture was adjusted to 6.2 with Cl and the batch was subsequently refined with pure water.
0 ml. A sample for measuring lipolytic activity immediately from this batch (“Null-Minuten-p
and the remaining batch was placed in a water bath at 30 ° C.
Incubated. Mix the batch thoroughly for sampling,
The sample is removed using a suitable pipette, immediately diluted with ice-cold lipase solvent and used for 8 to 16 minutes for lipase measurement.
0.5 ml and 1.5 with lipolytic activity of FIP-E
The test solution between ml was made available. Examples of the lipase solvent include FIP / Ph. Eur. Purified water 9 according to
10.0 g of NaCl in 00 ml, tris- (hydroxymethyl) -aminomethane (hereinafter "Tris (TRI
S) ") 6.06 g and maleic anhydride 4.9
g of the solution was used, the pH value was adjusted to pH 7 with 4N sodium hydroxide solution and the solution was made up to 1000 ml with pure water.

To check the time course of the lipase activity, further samples were taken after 15 minutes, 30 minutes, 60 minutes, 120 minutes and 180 minutes from the batch with thermostat and in each case within 30 minutes the lipase activity The activity was determined by FIP / Ph. Eur. (Lauers, p. 78).

"Null Minute Sample-
Probe), the lipase activity in FIP-E / ml as 100% value and the activity value measured during other incubation times as a percentage of this value. The result is a second. It is listed in the table.

1.2. Lipase stability with lecithin addition
Soybean lecithin (Roth) for the production of test lecithin solutions
(100% phospholipid content of the company) was kneaded with a small amount of pure water at room temperature, and then made up to 20.0 ml. The mixture is sonicated with stirring for about 2 minutes until a homogeneous colloidal solution is achieved. Next, 54694FIP-E / g
80.0 mg of water-soluble lyophilized F-pancreatin having lipolytic activity is dissolved in 4.0 ml of pure water,
The pH value in the solution was adjusted to 6.2 with 1N HCl. 0.4 ml of lecithin solution (lecithin 2.5%, based on F-pancreatin powder used) was added and the batch was brought to 5.0 ml with ice-cold deionized water with thorough mixing.

The determination of the lipolytic activity with respect to the sampling and the respective incubation time is described in 1.1. Performed as described in. The results are given in Table 2.

Table 2: Changes in lipolytic activity in aqueous pancreatin solution with or without lecithin addition

[0058]

[Table 2]

2. Aqueous pancreas over a period of 8 hours
Stabilization of Lipolytic Activity of Chin Formulations Loss of lipolytic activity occurs in aqueous pancreatin formulations depending on various factors such as temperature, pH value and proteolytic activity of the contained protease. By the addition of complex lipids such as lecithin, lipase activity can be clearly stabilized during incubation at 25 ° C. for 8 hours. Thus, in the following experiments, the lipolytic activity in aqueous suspensions and aqueous solutions of F-pancreatin with or without the addition of complex lipids in each case for 8 hours was compared.

2.1 For the production comparison of the incubating batches, clear pancreatin solutions and suspensions of F-pancreatin with and without lecithin addition were tested in each case.

The following aqueous formulations were prepared: a) Pancreatin solution without lecithin Lyophilized water-soluble F-pancreatine powder 77.5
mg in ice-cold pure water as described in 1.1,
The pH was adjusted to 6.2 with 1N HCl.

B) Pancreatin solution with lecithin Lyophilized water-soluble F-pancreatin powder 81.0
After adjusting the pH value to 6.2 as described in 1.2 to 0.9 mg, 0.94 ml of a lecithin solution was added, and the mixture was made up to 5.0 ml with ice-cold pure water.

C) Lecithin-free pancreatin suspension 2.0 g of F-pancreatin was added to 100 ml of ice-cooled pure water.
For 30 minutes. A cloudy suspension was obtained.

D) Pancreatin suspension with lecithin F-pancreatin 2.0 g soy lecithin 100 mg
(Roth) and add 30 ml in 100 ml of ice-cooled pure water.
Stirred for minutes. A cloudy suspension was obtained.

2.2 Performing the experiment A sample for measuring the lipolytic activity was taken from the ice-cooled solution or suspension prepared in 2.1 immediately after its preparation ("Null-Minuten-Prob").
e "). The remainder of the batch was then placed in a test tube for 8 hours 2
Incubate at 5 ° C. During this time, a further sample was taken after 30 minutes and subsequently every hour. Sampling, dilution and measurement of lipase activity are in principle 1.1.
The experimental temperature was 25 ° C., different from the above instructions.

The lipase activity (expressed in FIP-E / ml) of the "zero-minute sample" of the incubation batch is taken to be the 100% value, and the activity value measured during the incubation time is taken as a percentage of this value. The results are shown in Tables 3 and 4 below.

Table 3: Course of lipase stability over 8 hours in pancreatin solution with or without lecithin addition

[0068]

[Table 3]

Table 4: Course of lipase stability over 8 hours in pancreatin suspension with or without lecithin addition

[0070]

[Table 4]

3. Microbial protea by addition of complex lipids
Stabilization of microbial lipase against protease In the following experiments, it was shown that the activity of microbial lipase in the presence of active microbial protease can also be stabilized by the addition of complex lipids. For this purpose, three test solutions were prepared containing microbial lipase, microbial lipase + microbial protease and microbial lipase + microbial protease with lecithin addition. Continued
The lipolytic activity in these test solutions was measured and compared to each other by means of a table.

3.1 Preparation of test solutions The following solutions were prepared: a) Lipase solution Rhizopus oryzae lipase with activity of 170000 FIP-E / g (Lipase 7-AP15, Amano P)
Pharmaceutical Co., Ltd. , LTD Nagoya, Japan) 245 mg was dissolved in 50 ml of a 1% ice-cold sodium chloride solution.

B) Protease solution Aspergillus protease having an activity of 7600 FIP-E / g
case) (Prozyme 6; AmanoPharm)
Aceutical Co. , LTD Nagoya, Japan)
390 mg in 1 ml of ice-cold sodium chloride solution 25 ml
Was dissolved.

C) Lecithin solution Lecithin (Soja-Reinlecit from Roth)
hin, phospholipid content about 98%) in pure water (Ba)
rnstead's ^"Nanopure (R)") 40
The mixture was dissolved in ultrasonic waves for about 2 minutes while shaking to form a colloid.

From these solutions, the following total volume 5
ml of incubation solution were prepared: Table 5: Rhizopus-incubation solution for measuring lipase activity

[0076]

[Table 5]

The pH values of all incubating solutions are 37
It was 6.5 at ° C.

3.2 Implementation of Experiment  Preparation from ice-cold incubation solutions I, II and III
Immediately thereafter, a sample for measuring the original activity (“Null M
inten-Probe ") and take out three
The litter residue was incubated at 37 ° C. in a water bath in a test tube.

For sampling, the batch was mixed well and the sample was removed with a suitable pipette and immediately diluted with ice-cold lipase solvent as described in 1.1. A specific amount was taken from each of these sample solutions (indicated by "X" in Table 6) and diluted to 5 ml with a 1% sodium chloride solution. 0.5 ml each time from these test solutions
Was used in the lipase activity test.

Table 6: Amount of sample removed for test solution production

[0081]

[Table 6]

3.3 Rhizopus-
Determination of lipase activity The contact activity of Rhizopus-lipase was determined by measuring the amount of free fatty acids produced from olive oil emulsions in the presence of 0.025% sodium taurocholate at pH 7.0 and at a defined time at 37 ° C. Subsequent titrations were performed to pH 9.0 to ensure that all fatty acids were detected. Blind measurement of the titration of the substrate emulsion was used to identify titratable substances not generated by lipase activity.

The Rhizopus-lipase activity of a test substance is determined by the rate at which a suspension of the substance hydrolyzes an olive oil emulsion and the rate at which a suspension of a Rhizopus-lipase reference standard hydrolyzes the same olive oil emulsion under the same conditions. Was determined by comparison.

Reagent 1. Water: Barnstead's pure water “Nanopu”
re ^(R) "was used. In the following," pure water "is indicated.

[0085] 2. Gum arabic solution: gum arabic 11
0 g and 12.5 g of calcium chloride were dissolved in pure water with stirring (about 3 hours), made up to 1000 ml and centrifuged. The solution was transferred to a 250 ml plastic container and stored at -20 <0> C.

Gum arabic (Acaciae-Gumm)
i, DAB 10 / Ph. Eur. ), Calcium chloride p. a. 3. Substrate emulsion: 130 ml of olive oil and 400 ml of gum arabic solution (2) were emulsified in a suitable stirrer at high rpm for 15 minutes. The temperature was kept below 30 ° C. At least 90% of the droplets in the emulsion are 3
None had a diameter smaller than μm and none was larger than 10 μm. The emulsion was made fresh daily. Olive oil (stored in refrigerator), DAB quality.

[0087] 4. Sodium taurocholate solution 0.5
% ( M / V): 0.5 g of sodium taurocholate (mixture of lipase activation mixture, conjugate bile acid, especially sodium taurocholate) was dissolved in 100.0 ml with the purest water. Solutions were prepared fresh daily.

Sodium taurocholate (lipase activation mixture), FIP. 5. Sodium chloride solution 1% of the most pure water (m / V): 6. Buffer pH 4.5: 2 g of sodium chloride and 9.2 g of sodium dihydrogen phosphate are dissolved in about 950 ml of pure water, adjusted to pH 4.5 with hydrochloric acid, and 1000 m with pure water.
l.

Sodium chloride p. a. , Sodium dihydrogen phosphate, NaH ₂ PO ₄ × H ₂ O.

[0090] 7. Caustic soda solution 0.1N: Reference suspension: Rhizopus-lipase control standard is stirred into buffer (6), sodium chloride solution (5) and enzyme solution is 12-18 units of Rhizopus-lipase. Diluted to contain active FIP-E / ml. 63 mg for a standard with 55000 FIP-E / g in 20 ml of buffer (6) in an ice bath
Dissolved in minutes. 10 ml of this solution was diluted to 100 ml in an ice bath with sodium chloride solution (5). This solution
5 ml was used for the test.

Rhizopus-lipase control FIP (fungal lipase, FIP standard).

Test solutions: Test solutions I, II and III
It was used.

Conducting the Experiment: The automatic titration device of the titration system "pH-Stat" from Radiometer, Inc.
The end point of the -Stat-titration was adjusted to pH 7.0. The following were added to the reaction vessel: olive oil emulsion, FIP (3) 12.0 ml pure water (1) 6.5 ml sodium taurocholate solution (4) 1.0 ml The mixture was brought to 37.0 ° C. The pH value was adjusted to pH 7.0 with 0.1N sodium hydroxide solution (7). Thereafter, the biuret was adjusted to "0".

The reaction was started by adding 0.5 ml of the reference suspension if a reference value was to be measured or by adding 0.5 ml of the test solution if lipase activity in the test solution was to be measured. After 10 minutes, the endpoint of the pH-Stat-titration was adjusted to pH 9.0. When the pH reached 9.0 (this process took less than 30 seconds), the titration was interrupted,
The consumption of 0.1N caustic soda solution was read.

To confirm the blind values, the end point of the titration in the titrator for the reference suspension and the test solution was immediately adjusted to pH 9.0. PH value in reaction batch
After manual adjustment to 7.0 and after addition of 0.5 ml of reference suspension or test solution, titration was immediately carried out to pH 9.0. The Rhizopus-lipase activity in FIP-E / ml was calculated from the read consumption of 0.1N sodium hydroxide solution.

The original lipase activity in FIP-E / ml determined in test solutions I, II and III was 10
The value was set to 0%. The activity values measured during the next one-hour incubation period were then each time relative to this starting value and are reported in Table 7.

Table 7: Stabilization of Rhizopus-lipase activity by soy lecithin

[0098]

[Table 7]

4. Examples of pharmaceutical preparations A) 20.0 g of F-pancreatin are taken up in 400 ml of water and the pH is quickly adjusted to 6.2 by addition of 1N HCl. Extract under these conditions at 4 ° C. for 1 hour. The clear supernatant containing the pancreatin extract was
Centrifuge at 0 g and subsequently sterile filter through a 2 μm pore size filter.

B) 1.0 g of soy lecithin (98% phosphatidylcholine) is dissolved in 50 ml of water and sterilized in a sealed glass ampoule at 140 ° C. for 45 minutes.

C) 400 ml of the pancreatin extract prepared in A) are mixed with 25 ml of the lecithin solution prepared in B), cold and under aseptic conditions. The mixture is freeze-dried, whereby 16 g of a powder are obtained, which is transferred under aseptic conditions to 2.5 g portions into a 100 ml leach flask.

5. Examples of kits for pharmaceutical applications 5.1. Kit 1 A) Take 40.0 g of F-pancreatin in 400 ml of water and adjust the pH rapidly to 6.2 by adding 1N HCl. Extract at these conditions for 1 hour at 4 ° C. The clear supernatant containing the pancreatin extract was
Centrifuge at 0 g and subsequently sterilize with a filter having a pore size of 2 μm. Extract is 25m under sterile conditions
Transfer to a 100 ml leach flask and freeze-dry.

B) 2.0 g of soy lecithin (98% phosphatidylcholine) is dissolved in 20 ml of water and homogenized by means of ultrasound. The colloidal solution is sterilized in a sealed glass ampoule at 140 ° C. for 45 minutes. From now on 1
Remove 2.5 ml aliquots, mix with 1600 ml of sterile water and transfer 100 ml aliquots to leach flask.

For application, the contents of the solid flask obtained in A) are dissolved in the contents of the flask of the solution obtained in B).

5.2. Kit 2 A) 40.0 g of F-pancreatin are taken in 800 ml of water and the pH is quickly adjusted to 6.2 by addition of 1N HCl. Extract at these conditions for 1 hour at 4 ° C. The clear supernatant containing the pancreatin extract was
Centrifuge at 0 g and discard the precipitate.

B) 32.0 g of mannitol was added to 200 ml of water
And the pancreatin extract 800 obtained in A)
and the combined solution is sterile filtered.

C) 5.0 g of soybean lecithin (98% phosphatidylcholine) is dissolved in 50 ml of water and homogenized by means of ultrasound. The colloid solution is sterilized in a sealed glass ampule at 140 ° C. for 45 minutes.

D) 1000 ml of the pancreatin extract obtained in B) is mixed with 16 ml of the lecithin solution obtained in C) under aseptic conditions and freeze-dried. The resulting powder is subdivided into 10.0 g and aseptically transferred to a 200 ml flask.

E) Deionized and sterile filtered water was added to 20
Transfer to a 0 ml flask under aseptic conditions.

For the application, the contents of the flask of the powder obtained in D) are dissolved in the contents of the flask of water transferred in E).

Claims (21)

[Claims] 1. Lipolysis under the action of water in a water-soluble pharmaceutical preparation of a lipase and protease-containing digestive enzyme mixture, which is suitable for the production of an aqueous solution for continuous introduction into the gastrointestinal tract using a probe. Use of complex lipids as stabilizing additives for reduced activity. 2. The digestive enzyme mixture containing lipase and protease is a digestive enzyme mixture containing pancreatin.
Use of the complex lipid according to claim 1. 3. A plant lipase, in addition to pancreatin,
3. The composite lipid of claim 2, which is used as a stabilizing additive to a pharmaceutical formulation of a pancreatin-containing digestive enzyme mixture, comprising an additional lipase selected from the group consisting of bacterial lipase and lipase from a fungal culture. use. 4. The use of the complex lipid according to claim 1, wherein a phospholipid, a glycolipid, or a mixture thereof is used as the complex lipid. 5. The use of the complex lipid according to claim 4, wherein a phospholipid or a lipid mixture containing the same is used as the complex lipid. 6. A compound of the general formula I as a phospholipid [Wherein R ¹ represents hydrogen or a hydrocarbon group as the case may be.
Represents an alkanoyl group having 10 to 25 carbon atoms, which may contain 10 double bonds, and R ² represents 10 to 25 carbon atoms wherein the hydrocarbon group may optionally contain 1 to 4 double bonds.
Represents an alkanoyl group having 5 carbon atoms, or, if R ¹ does not represent hydrogen, it can also represent hydrogen, and R ³ is hydrogen, amino, lower trialkylammonium, a carbon atom having an amino function. Represents a lower alkyl group optionally substituted by a bonded carboxyl group or a cycloalkyl group substituted by hydroxy, wherein R ⁴ is hydrogen or 10 to 25 carbon atoms which may contain 1 to 4 double bonds; Represents a hydrocarbon chain having an atom, and A represents oxygen or NH]. The use of a complex lipid according to claim 5, wherein a salt of an anion with a physiologically acceptable cation or a mixture thereof is used. 7. The complex lipid according to claim 6, wherein the group R ³ in the phospholipid of the general formula I is selected from the group consisting of hydrogen, aminoethyl, cholyl, ceryl, glyceryl or myo-inosityl. Use of. 8. The complex lipid according to claim 7, wherein a phospholipid from phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinosit and sphingomyelin or a lipid mixture containing these is used. Use of. 9. Use of the complex lipid according to claim 4, wherein soybean, rapeseed, corn, sunflower, peanut, chicken egg or lecithin from a microorganism is used as the complex lipid. 10. Use of the complex lipid according to claim 9, wherein lecithin from soybean is used as the complex lipid. 11. The method according to claim 11, wherein the lipids are added to the pancreatin used in the pharmaceutical formulation, already during its production or during processing for stabilization against a reduction in lipolytic activity during the step combined with water treatment. The use of the complex lipid according to any one of claims 2 to 10, wherein the complex lipid is added. 12. A water-soluble pharmaceutical preparation of a digestive enzyme mixture containing lipase and protease, which is suitable for preparing an aqueous solution for continuous introduction into the gastrointestinal tract by sonde application. 11. An amount sufficient to stabilize the degradation activity against reduction.
A water-soluble pharmaceutical preparation of a mixture of a lipase and a protease-containing digestive enzyme, comprising the complex lipid according to any one of the above. 13. The pharmaceutical preparation according to claim 12, wherein the lipase and protease-containing digestive enzyme mixture is a pancreatin-containing digestive enzyme mixture. 14. The pharmaceutical preparation according to claim 13, wherein the digestive enzyme mixture contains, in addition to pancreatin, plant lipase, bacterial lipase and lipase from a fungal culture. 15. The lipolytic activity is stabilized by the aqueous solution of the formulation, and the lipolytic activity of the formulation contains an amount of the complex lipid such that the lipolytic activity of the aqueous solution is reduced by up to 20% over a period of 8 hours. Item 15. The pharmaceutical preparation according to any one of Items 12 to 14. 16. The lipolytic activity of the aqueous solution of the preparation is 24.
16. The pharmaceutical formulation according to claim 15, characterized in that it contains an amount of the complex lipid which decreases by up to 50% over time. 17. The method according to claim 12, wherein the complex lipid is contained in a concentration of 1 to 10% by weight with respect to the digestive enzyme mixture for stabilization against reduction of lipolytic activity. The pharmaceutical preparation according to claim 1. 18. The pharmaceutical preparation according to any one of claims 12 to 17, wherein the pancreatin contained is pancreatin pretreated autolytically. 19. To produce an aqueous solution of a digestive enzyme mixture without bacterial growth, stabilized against reduced lipolytic activity, suitable for continuous introduction into the gastrointestinal tract using a probe. 11. The kit according to claim 1, which may comprise as components: a) a complex lipid according to any one of claims 1 to 10, which is optionally sufficient to stabilize the aqueous solution to be produced against a reduction in lipolytic activity. Lipase and protease-containing digestive enzyme mixture, water-soluble,
And b) a physiologically acceptable salt and auxiliaries in addition to water, which can contain physiologically tolerable salts and auxiliaries, and b) a lipolytically active substance in the solid pharmaceutical preparation mentioned in a). If the complex lipid is not present in an amount sufficient to stabilize the aqueous solution to be produced against the reduction, an additional amount of the lipid is required to stabilize the aqueous solution to be produced against the reduction in lipolytic activity. Item 11. An aqueous solution of a digestive enzyme mixture comprising an aqueous solvent free of bacterial growth in an amount sufficient for the production of an aqueous solution containing the complex lipid according to any one of Items 1 to 10. Kit to do. 20. The kit according to claim 19, wherein the solid preparation a) is a lyophilized lipase and protease-containing digestive enzyme mixture, optionally containing complex lipids. 21. The kit according to claim 20, wherein the freeze-dried digestion enzyme mixture contains pancreatin.